Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) is a cell surface protein belonging to the glypican family of heparin sulfate proteoglycans. GPC3 gene encodes and produces a core protein precursor of about 70 kDa, the precursor protein can be cleaved by furin into a soluble amino terminal (N-terminal) peptide of about 40 kDa which is capable of entering blood, and a membrane binding carboxyl terminal (C-terminal) peptide of about 30 kDa containing 2 heparin sulfate chains. GPC3 protein is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor.
GPC3 is highly expressed in fetal liver and not expressed in normal adult liver tissue, but its expression is reactivated in hepatocellular carcinoma, and has a very close association with the development of liver cancer, the detection rate of GPC3 expression is relatively high during early stage of liver cancer and increases along with the development of liver cancer. Meanwhile, the expression of GPC3 is not detected in liver adenocarcinoma, cholangiocarcinoma, liver metastasis and 12 common solid tumors and 21 non-hepatoma cell lines. Furthermore, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma and other tumors. Considering its specifically high expression in hepatocellular carcinoma, melanoma and other tumors, GPC3 is considered to be a candidate target for tumor immunotherapy.
There have been reports about liver cancer detection utilizing anti-GPC3 antibody and investigation programs on antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) using anti-GPC3 antibody. Antibodies for therapeutic uses generally recognize the C-terminus of the GPC3 protein. However, antibody therapy is restrained by the in vivo half-life of antibody in blood circulation, which is mostly less than 23 days. Therefore, antibody therapy for tumor requires continued administration and/or increased dosage of administration, which would result in increased treatment cost to the patients, and in certain circumstances, may even lead to unwilling termination of treatment. Moreover, as an exogenous protein, therapeutic antibody may be associated with in vivo risk of causing allergic reaction and generating neutralizing-antibodies against said therapeutic antibody.
The role of T lymphocytes in immune response against tumor is gaining more and more attention. Adoptive immunotherapy based on T lymphocyte has achieved certain effects on some tumors, and such immunotherapy may overcome the above limitations of antibody therapy, but its efficacy on most tumors is still unsatisfactory (Grupp S A, et al., Adoptive cellular therapy, Curr Top Microbiol Immunol, 2011, 344:149-72). In recent years, enlightened by the finding that specific recognition of CTL towards target cells is dependent on T lymphocytes receptor (T Cell Receptor, TCR), scFv antibody against tumor associated antigen is fused with intracellular signal activation motif of T lymphocyte receptor such as CD3ζ or FcεRIγ into a chimeric antigen receptor (Chimeric Antigen Receptor, CAR), and presented at the surface of T lymphocyte through genetic modification by lentiviral transduction or similar means. Such CAR T lymphocytes can selectively direct to tumor cells and specifically kill tumor in a MHC-independent manner, where MHC stands for major histocompatibility complex. CAR T lymphocyte therapy is a novel immunotherapeutic strategy in the field of cancer immunotherapy (Schmitz M, et al., Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer, J Biomed Biotechnol, 2010, doi: 10.1155/2010/956304).
Chimeric antigen receptor comprises an extracellular binding domain, a transmembrane region and an intracellular signaling domain. Generally, the extracellular domain comprises an scFv that is capable of recognizing a tumor-associated antigen, the transmembrane region employs the transmembrane region from molecules such as CD8, CD28 and the likes, and the intracellular signaling domain employs an immunoreceptor tyrosine-based activation motif (ITAM) CD3ζ or FcεRIγ and the intracellular signaling domain of co-stimulatory signaling molecule such as CD28, CD137, CD134 and the likes.
In the first generation CAR T lymphocyte, the intracellular signaling domain comprises ITAM only, and parts of the chimeric antigen receptor are connected in the form of scFv-TM-ITAM. Such CAR T can induce cellular cytotoxic effect against tumor, but the level of cytokines secreted is relatively low, and no sustaining anti-tumor effect could be induced in the body (Zhang T. et al., Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Can Res 2007, 67 (22): 11029-11036).
In the second generation CAR T lymphocyte that developed afterwards, an intracellular signaling domain of CD28 or CD 137 (also known as 4-1BB) is further included, and parts of the chimeric antigen receptor are connected in the form of scFv-TM-CD28-ITAM or scFv-TM-CD137-ITAM. Co-stimulatory effect of B7/CD28 or 4-1BBL/CD137 in the intracellular signaling domain induces sustained proliferation of T lymphocytes, and is capable of increasing the level of cytokines such as IL-2, IFN-γ and others secreted by T lymphocytes, as well as improving the in vivo survival period and the anti-tumor effect of the CAR T (Dotti G. et al., CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients J Clin Invest, 2011, 121 (5): 1822-1826).
In the third generation CAR T lymphocyte that developed in recent years, parts of the chimeric antigen receptor are connected in the form of scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, the in vivo survival and the anti-tumor effect of the CART is further improved (Carpenito C, et al., Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD 137 domains, PNAS, 2009, 106(9): 3360-3365).
Besides the attractive prospect of CAR T lymphocyte in tumor immunotherapy, some potential risks shall be taken into account. For instance, certain normal tissues may exhibit low expression of specific antigen to be recognized by the CAR, this may results in damage by CAR T lymphocytes to such normal tissues. For example, treatment against carbonic anhydrase IX, the antigen expressed in tumor cells of patients having renal cell carcinoma, is the first reported case of clinical application of adoptive therapy with CAR T lymphocytes, which is also the first case reporting on-target off-tumor effect of CAR T lymphocytes. After multiple administrations of CAR T lymphocytes, patients developed liver toxicity of grades 2-4. Upon analysis, the cause is believed to be the CAIX expression in a low level on bile duct epithelial cells, this clinical trial was discontinued while assessment about therapeutic outcomes in patients are excluded (Stoter G. et al., Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience, J din oncol, 2006, 24 (13): e20-e22; Ngo M C, et al., Ex vivo gene transfer for improved adoptive immunotherapy of cancer Human Molecular Genetics, 2011, R1_R7). Furthermore, the excessive co-stimulation signal in CAR may reduce the threshold required for activating effector cells, such that genetically modified T lymphocyte may be activated at conditions of rather low level of antigen or at the absence of antigen pulse, and resulting in the release of large amount of cytokines which may induce so-called “cytokine storm”. This signal leakage will cause off-target cytotoxicity, resulting in non-specific tissue damage. For example, sudden death of a patient caused by such “cytokine storm” induced by low Her2 expression in normal lung tissue was observed during a clinical treatment using a third-generation CAR T cells targeting Her2 for patients having advanced colorectal cancer with liver and lung metastasis (Morgan R A, et al., Report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2 Molecular Therapy, 2010, 18 (4): 843-851).
Therefore, a strong need exists in the art for a tumor treatment regimen using lymphocyte encoding GPC3-specific chimeric antigen receptor while overcoming the above defects.